An accelerated Rauhut–Currier dimerization enabled the synthesis of (±)-incarvilleatone and anticancer studies

The total synthesis of racemic incarvilleatone has been achieved by utilizing unexplored accelerated Rauhut–Currier (RC) dimerization. The other key steps of the synthesis are oxa-Michael and aldol reactions in a tandem sequence. Racemic incarvilleatone was separated by chiral HPLC and the configuration of each enantiomer was determined by single-crystal X-ray analysis. In addition, a one-pot synthesis of (±)-incarviditone has been achieved from rac-rengyolone by using KHMDS as a base. We have also assessed the anticancer activity of all the synthesized compounds in breast cancer cells nonetheless, they exhibited very limited growth suppression activity.


Introduction
(±)-Incarvilleatone (1) is a dimeric cyclohexylethanoid isolated by Zhang and co-workers [1] in racemic form from the Chinese plant Incarvillea younghusbandii (Figure 1). This plant is used in Chinese folk medicine to treat dizziness and anemia [1].
Zhang and co-workers [1]   (±)-Incarviditone (2) presents a new carbon-skeleton by being the first benzofuranone dimer connected by a C-C bond. The cytotoxicity of (±)-incarviditone (2) has been assayed against cell lines A549, LOVO, HL-60, 6TCEM, and HepG2 and displayed cytotoxicity against the HL-60 cell line with an IC 50 value of 14.8 mg/mL and against the 6T-CEM cell line with an IC 50 value of 22.2 mg/mL.
Lawrence et al. [3] and Tang et al. [4] have reported the synthesis of (±)-incarvilleatone (1) and (±)-incarviditone (2) via biomimetic dimerization of (±)-rengyolone (3). Despite of these syntheses, the synthesis of the dimeric natural product (±)-incarvilleatone (1), whose monomeric unit (±)-rengyolone (3) was connected by accelerated intermolecular Rauhut-Currier (RC) reaction was not reported in literature. Herein, we present the total synthesis of (±)-incarvilleatone (1) by utilizing an unexplored accelerated Rauhut-Currier (RC) dimerization as a key step. In addition to that HPLC separation of (±)-incarvilleatone (1) into its enantiomers (−)-incarvilleatone (−)-1 and (+)-incarvilleatone (+)-1 and their X-ray crystallographic analysis which has not yet been reported in the literature is described. The reaction was discovered by Rauhut and Currier [5] in the year 1963. It is a nucleophile-catalyzed C-C bond-forming reaction between two Michael acceptors. This reaction provides access to diverse classes of densely functionalized molecules. Rauhut-Currier (RC) dimerization has some limitations, such as its controlling selectivity for intermolecular reactions in differently activated alkenes, and low reactivity. Han and co-workers [6] addressed the latter one by designing a substrate in which nucleophile functionality is also present in the Michael acceptor to accelerate the reaction. In conventional intermolecular RC reactions, the reaction proceeds by the intermolecular addition of a nucleophilic catalyst to the enone substrate to generate an enolate in the first step. In the second step the enolate ion attacks the other Michael acceptor at β-position in an intermolecular fashion to form a C-C bond between the two Michael acceptors.
This whole process involves two intermolecular conjugate additions, which leads to low reactivity. In case of intramolecular RC reactions, a high reactivity is observed. This is due to one intermolecular and one intramolecular conjugate addition reaction involved. The low reactivity of intermolecular RC reactions can be improved by incorporating the nucleophilic functionality within a molecule like I (Scheme 1). Scheme 1: Possible modes of accelerated intermolecular RC reaction, drawn according to [6].
This nucleophilic functionality present within the enone system first undergoes an intramolecular conjugate addition and is followed by an intermolecular conjugate addition to form a C-C bond (path A). In an alternative approach (path B) first I undergoes a nucleophilic conjugate addition in intermolecular fashion to give intermediate III and followed by an intramolecular addition to give compound IV. In both paths, the involvement of one intramolecular and one intermolecular conjugate addition reaction leads to notable high acceleration in RC reactions.

Scheme 3:
Synthesis of RC dimerized product (±)-4. accelerated intermolecular Rauhut-Currier reaction. The presence of a nucleophilic functionality (hydroxy group) and an enone system within the same molecule are needed to accelerate the intermolecular RC reaction.
The yield of this reaction could not be improved even by prolonging the reaction time (up to 24 h). However, when we treated (±)-rengyolone (3) with 1.0 M TBAF (2 equiv) in THF at room temperature, we obtained the dimeric product (±)-4 in 41% yield. The formation of dihydroxy dimerized RC product (±)-4 was confirmed by NMR spectra. In the 1 H NMR spectrum, the olefin proton was observed at δ 6.75 ppm as a singlet, and the two hydroxy protons were observed at δ 5.60 (s, 1H) and 5.03 (s, 1H) ppm, respectively. In the 13 C NMR, the two carbonyl groups appeared at δ 197.4 and δ 209.4 ppm, and the corresponding two olefinic carbons were observed at δ 135.7 and δ 148.5 ppm. The formation of the dihydroxy compound (±)-4 was also confirmed with a D 2 O shake experiment. When we added a drop of D 2 O to the 1 H NMR sample, the peaks corresponding to the two hydroxy groups were completely absent at δ 5.60 (s, 1H) and 5.03 (s, 1H) ppm ( Figure S1, Supporting Information File 1). The formation of the dihydroxy dimeric compound (±)-4 was further confirmed by HRMS, which showed a peak at 331.1150 corresponding to the C 16 H 20 O 6 Na [M + Na] + ion. After some efforts, to our delight, we could obtain single crystals of (±)-4 using EtOAc as a solvent. Finally, the formation of heterodimerized dihydroxy RC product (±)-4 was confirmed by single-crystal X-ray analysis [16]. It is perti-Scheme 4: Proposed reaction mechanism for the formation of compound (±)-4 under TBAF-mediated Rauhut-Currier reaction. nent to mention here that in this reaction we obtained the heterochiral dimerized product (±)-4. We did not observe any homochiral dimerized product formation under TBAF reaction conditions. A plausible mechanism for the formation of heterochiral dimerized dihydroxy RC product (±)-4 through accelerated RC [6] is outlined in Scheme 4.
However, when we treated compound (±)-4 with a strong base such as KHMDS (2 equiv) in THF at 0 °C (Table 1, entry 7), we obtained the product as a colorless solid (15% yield) after 24 h stirring at room temperature (Scheme 5).

Scheme 6:
Separation of rac-incarvilleatone (1) and determination of absolute configurations of both the enantiomers using single crystal X-ray analysis.
A previous study [1] showed that (±)-incarviditone (2) has a limited anticancer activity. The anticancer activity is closely correlated with a growth suppression. Colorimetric MTT assays are widely used to examine a growth suppression. In these assays, viable cells reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to its insoluble formazan by oxidoreductase enzymes in a nicotinamide adenine dinucleotide phosphate (NADPH) dependent manner. Thus, reduction of MTT is closely related to the viability of the cells. Therefore, we have examined the growth-suppressive effect of all the synthesised compounds using the MTT assay [18][19][20].
The results revealed that the investigated compounds have a very minimal effect on the growth of the breast cancer cell line MCF7. For instance, we did not observe the 50% growth inhibition event at 100 µM. In contrast, 5-fluorouracil potently inhibited the growth of MCF7 cells with 50% growth inhibition at 7.1 ± 0.62 µM ( Figure 2).

Conclusion
In summary, we have successfully achieved the total synthesis of (±)-incarvilleatone (1) starting from rac-rengyolone (3) through accelerated RC intermolecular dimerization catalyzed by TBAF to synthesize a heterochiral dimerized product (±)-4, followed by a one-pot oxa-Michael and aldol reaction sequence using KHMDS as a base. The synthesized (±)-incarvilleatone (1) was separated into its individual enantiomers by using HPLC (analytical Chiralpak IA column). The absolute configurations of both the enantiomers were determined by single crystal X-ray analysis [16]. We have also synthesized (±)-incarviditone (2) starting from rac-rengyolone (3) by using KHMDS as a base. The antiproliferative activity of these compounds was tested using MTT assays and the results revealed that these compounds are less efficient in inhibiting the growth of breast cancer cells.